CD14+ monocytes were isolated from healthy donor peripheral blood enriched leukopacks (Blood Bank, National Institute of Health, Bethesda, MD, USA) using MACS CD14 MicroBeads (Miltenyi Biotec, Auburn, CA, USA). Monocytes were differentiated into macrophages using 25 ng/ml M-CSF (R&D Systems, Minneapolis, MN, USA) (M-Mac) or a combination of M-CSF and 100 ng/ml IL-27 (R&D Systems) (I-Mac). Initially the monocytes were in Macrophage serum free media (Invitrogen, Carlsbad, CA, USA) which was replaced after three days with DMEM (Invitrogen) with 10% FBS (HyClone Laboratories, Logan, UT, USA), 25 mM HEPES (Quality Biology, Gaithersburg, MD, USA) and 5 µg/ml Gentamicin (Invitrogen) containing the same concentration of IL-27. Cells were incubated at 37°C in a 5% CO2 incubator for a total 7 days.
Extracted molecule
total RNA
Extraction protocol
Cells were collected in RLT buffer. Total RNA was isolated using Qiagen Rneasy kit. cDNA synthesis and amplification used Ambion WT expression kit.
Label
Biotin
Label protocol
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix)
Hybridization protocol
Samples were hybridized using Affymetrix hybridization kit materials • Heat cocktails at 99° for 5 minutes, then 42° for 5 minutes centrifuge at max speed for 1 minute (N.B. this deviates from Affy SOP). • Transfer 200μl of hyb solution to each array, then tape holes and parafilm. • Hybridize 16 hours at 45° at 60rpm• Fluidics washing is FS450_0001
Scan protocol
Affymetrix Gene ChIP Scanner 3000 7G
Data processing
Data were processed using Partek software. RMA background correction with quantile normalization probe group file: HuGene-1_0-st-v1.r4.pgf meta-probeset file: HuGene-1_0-st-v1.r4.mps
